Microbial diversity and proxy species for human impact in Italian karst caves

To date, the highly adapted cave microbial communities are challenged by the expanding anthropization of these subterranean habitats. Although recent advances in characterizing show-caves microbiome composition and functionality, the anthropic effect on promoting the establishment, or reducing the presence of specific microbial guilds has never been studied in detail. This work aims to investigate the whole microbiome (Fungi, Algae, Bacteria and Archaea) of four Italian show-caves, displaying different environmental and geo-morphological conditions and one recently discovered natural cave to highlight potential human-induced microbial traits alterations. Results indicate how show-caves share common microbial traits in contrast to the natural one; the first are characterized by microorganisms related to outdoor environment and/or capable of exploiting extra inputs of organic matter eventually supplied by tourist flows (i.e. Chaetomium and Phoma for fungi and Pseudomonas for bacteria). Yet, variation in microalgae assemblage composition was reported in show-caves, probably related to the effect of the artificial lighting. This study provides insights into the potential microbiome cave contamination by human-related bacteria (e.g. Lactobacillus and Staphylococcus) and commensal/opportunistic human associated fungi (e.g. Candida) and dermatophytes. This work is critical to untangle caves microbiome towards management and conservation of these fragile ecosystems.

www.nature.com/scientificreports/ been investigated in detail. Therefore, the consequence of tourist flows and human activities in general on microbial assemblages composition remains largely unknown.
To address this knowledge gap, our study intends to provide novel comprehensive insights into the microbial diversity and composition of 5 caves that include 4 show-caves and 1 recently discovered natural cave, displaying different environmental and geo-morphological conditions across the Italian peninsula. Through taxonomic comparison, we have characterized the principal microbial traits found in anthropized and pristine habitats, starting to shed light on potential microbial taxa that may be considered as indicative of human impact.

Results
Microbial community composition in cave sediments. The ITS1 dataset generated 5,793,980 raw sequence reads, resulting in 5,458,895 gene quality-filtered reads, ranging from 1252 up to 540,803 per sample. After singletons and rare taxa (< 5 reads) removal (1108 out of 10,595 ASVs total), a total of 9176 high-quality ASVs were obtained (Table S1). A total of 5,453,881 raw reads were generated from 16S rDNA dataset and accounted for a total of 4,806,902, which were grouped into 31,878 ASVs (out of a total of 65,037 ASVs) after quality filtering, with sequencing depths between samples ranging from 2066 to 265,442 reads .
Subsequently, the total 16S dataset was splitted by grouping the bacterial (31,015 ASVs) and archaeal (863 ASVs) ASVs separately for the downstream analyses (Table S2; Table S3). The 18S dataset was processed through a specific evaluation, extracting and analyzing only the sequences related to the microalgal component. A total of 97 ASVs were mapped for a total of 10,084 quality filtered reads, spread over 4 different groups (Alveolata, Chloroplastida, Chromista and Stramenopiles), using the Class taxonomic rank as the threshold for taxonomic identification as a consequence of the weak accuracy in the assignment towards higher ranks (Table S4).
Fungal community composition. In all caves, the dominant phylum was Ascomycota ( Fig. 2A) (ranging from 60% in Costacalda to 76.25% in Pertosa Auletta), followed by Basidiomycota and Mortierellomycota. A broad different distribution of fungal taxa pattern emerged between the four show -caves and the wild cave ( Fig. 2A,B,C,D). Notably, the genus Mortierella (Fig. 2D) was particularly abundant in the Costacalda wild cave (up to 31.8%), while in the show-caves did not exceed 10% (from 2.31% in Caudano up to 9.30% in Vento). The opposite was observed for the genus Candida (class Saccharomycetes), which was more abundant in the tourist caves (ranging from 13.36% in Caudano to 37.32% in Vento). Also, the genus Dipodascus was mainly present in the anthropized settings, even if at much lower extent compared to Candida, ranging from 0.14% in Pertosa-Auletta to 4% in Bossea. The genus Archaeorhizomyces (class Archaeorhizomycetes) was reported in show-caves     www.nature.com/scientificreports/ Archaeal community composition. Archaea showed 2 main compositional trends describing the archaeome throughout the investigated caves ( Fig. 4A,B,C,D). The first group is recurrent in all investigated caves and includes Thaumarchaeota and Euryarchaeota as the most abundant phyla, with the genera Nitrososphaera (ranging from 21.38% in Bossea to 41.35% in Pertosa-Auletta caves), Nitrosopumilus (between 14.23% and 41.76% in Bossea e Pertosa-Auletta) and Methanomassiliicoccus (ranging from 3% in Pertosa-Auletta to 21.35% in Costacalda). The same distribution across all caves was observed for taxa in the phylum Woesearchaeota, even if far less represented. The second group included taxa more frequent in show -caves as for the genera Acidianus (Phylum Chrenarcheota, class Thermoprotei, ranging from 2.62% Bossea to 11.34% in Pertosa-Auletta), Ignicoccus (average in show-caves 2.04%) and Thermocladium (4.14% highest value in Pertosa-Auletta); the genera Methanothermobacter and Methanobacterium (Phylum Euryarcheota, class Methanobacteria) were also found mainly in show -caves, the first with an average of 2.33% and the second with a frequency ranging between 0.55% and 3.9%. Other less abundant genera such as Methanosphaera and Thermococcus could be included in this second group.
The distribution of taxa detected for the phototrophic assemblage (Fig. 6) was extremely polarized between caves. One pole was represented by Costacalda Cave for which no unique ASVs were recorded. The other one was the cave of Pertosa-Auletta which accounted for 28 unique ASVs (28.9%), mostly belonging to the Chlorophyceae (39%), Chrysophyceae (32%) and Diatomea (18%) classes.
Human related microbial taxa. Due to the high abundance of some common human-related microbial taxa found in the show -caves investigated (i.e. Malasseziomycetes class and Candida genus for fungi, Lactobacillus and Bifidobacterium genera for bacteria), we decided to explore the occurrence of less abundant guilds too as potential proxies for direct human impact.
Furthermore, even the Clostridium genus (from 0.5% in Caudano to 1.6% in Bossea, vs. 0.07% in Costacalda; p > 0.05) seemed to exhibit a higher occurrence across the anthropized settings.

Discussion
The effects of alterations occurring on structure, biodiversity, microclimate, energetic condition 11,12 during the conversion of a natural cave into a show-cave are still almost unexplored. However, the increasing anthropization of new hypogean sites associated with human exploitation constantly impacts cave microbiomes, making their composition and functionalities unpredictable for future conservation and management 9,10,13,20 .
Herein, we deeply investigate, for the first time, microbiomes of four Italian show -caves using next generation sequencing approaches . We provided a first attempt to identify potential human-induced alterations by comparing show -caves with a recently discovered natural cave. The extensive sampling performed, together with the amplicon-sequencing analysis, led to a wide identification of microbial diversity, encompassing 17 phyla, 52 classes and 554 genera for the ITS dataset. On the other hand, the 16S barcode recorded 37 and 7 phyla, 73 and 8 classes, 1,339 and 49 genera for Bacteria and Archaea domains, respectively. For algae , a total of 4 phyla and 9 classes were identified; this low diversity of the phototropic counterpart is likely due to the condition of darkness characterizing the ground sampling area, although abundant algal growths were reported at the level of the rock formations in the examined show -caves 21 .
The mycobiome composition and the distribution of unique fungal ASVs across the 4 surveyed show -caves appeared more complex and diversified than those observed in the natural one. The predominance of Candida www.nature.com/scientificreports/ genus, which includes many species known as common colonizers of humans, in show -caves may reflect a relation with touristic flow. Candida has also been reported as an oligotrophic multi-stress tolerant fungus, skilled to colonize a wide range of natural, urbanized and human environmental contexts 22,23 . Cave visitors, as previously reported, may act as primary vector in the spreading of this genus, by bio-aerosols formation (breathing, sneezing or coughing), surface contact and shedding of skin scales, hair and lint from clothing 24 . Potential direct contamination by human-related species 25 of cave mycobiome are further supported by the higher abundance of dermatophyte fungal genera within tourist caves (e.g. Cutaneotrichosporon and Trichosporon). Conversely, the mycobiome of the natural cave was dominated by taxa belonging to the genera Mortierella and Cephalotricum, which include psychrotolerant cellulose-degrading fungi 26,27 , and by the genera Rodentomyces and Gymnoascus, which include psychrotolerant, coprophilic and keratinolytic species 28,29 , that may be related to the main caves macro-fauna components, i.e. rodents and bats. Furthermore, fast-growing saprophytic fungal genera (Penicillium, Chaetomium and Humicola) were also reported, even if in lower relative abundance. Such specific fungal traits have been reported as early wild cave colonizers 30 , able to cope with the constraining low-temperature and oligotrophic conditions in these habitats. On the other hand, within the 4 investigated show -caves, the notable abundance of Agaricomycetes (and generally Basidiomycota) may be the result of increasing ventilation during cave opening. In fact, airborne fungal spore contamination is facilitated by the atmospheric continuum between the outdoor and underground environment 31,32 . Moreover, the high occurrence of fastgrowing and heavily-sporulating fungi (Aspergillus and Penicillium), or common competent degraders and plant pathogens (Chaetomium, Phoma, Sarocladium and Dipodascus) could be the result of combined effects of tourist flows, spreading fungal alien species and extra complex organic matter input, and the implement of nutrients due to the consumption of food and beverages during events hosted in caves 33,34 . Finally, artificial lighting may have promoted the growth of lichenized fungi (Lecanoromycetes) inside the caves, as reported in previous studies 18,35 . In particular, their colonization of carbonatic rock substrate and speleothems may result in severe aesthetic and structural damage, due to the large extent of thalli and the high lichen acids production. The genus Tetracladium was always present in all caves, even if at low frequency. It belongs to the group of "Ingoldian fungi" or aquatic hyphomycetes, phylogenetically diverse fungi growing on decaying leaves and plant litter in streams 36 ; their presence in our environmental samples may be related to the presence of water flows present in all caves.
The compositional similarity found throughout the dataset for the Bacteria and Archaea domains, reflects their paramount role in biogeochemical cycles of caves biosphere: C, N, CH 4 -genesis and metabolism and S oxide-reduction, sustaining trophic networks and peculiar caves' geochemical processes, even in these deeply impacted habitats. In fact, a wide range of polymers degraders particularly adapted to cope with low temperature and oligotrophic conditions were found. For instance, Acidobacteria-subgroups (37% of the shared ASVs www.nature.com/scientificreports/ among the caves), Sphingomonas, Lysobacter, Polaromonas and a few widespread Mn-Fe oxidizing bacteria, such as family Planctomycetaceae members, Hyphomicrobium (Bacteroidota), Flavobacterium and Pseudomonas (Pseudomonadota) genera. Yet, the Pseudomonas high abundance reported in show -caves might be due to the indirect effects of human activities in these habitats. Extra nutrient inputs by tourist flows might stimulate fungal metabolic activity 37 and lead to high secretion of acids (i.e. oxalate and pyrophosphate), which in turn could trigger the metabolic activity and proliferation of Pseudomonas by chelating Mn 38 . Also related to the biogenic carbon cycle, the group of Methane-oxidizing bacteria (MOB) found along α,β,γ-Proetobacteria classes were widely present throughout the caves, underlining their key role in continuous CH 4 consumption in these subterranean ecosystems 39 . In contrast, Methanobacterium and Methanothermobacter were more present in tourist caves among the most abundant genera of the Archaea-methanogens counterpart. This compositional alteration could be the result of the indirect effects of tourist flows, which may increase indoor CO 2 levels up to 200% 40 , promoting the proliferation of these microorganisms, which use H 2 to reduce carbon dioxide molecules into methane. Compositional variations for the archaeal community have already been reported in touristic caves, due to anthropic pressure and after simulated organic matter treatment on cave microcosms 13,41 . The similar compositional pattern throughout the dataset of the main bacterial and archaeal players involved in the N cycle, i.e. Nitrospira (nitrite-oxidant), Gaiella (nitrate-reducing), Nitrososphaera and Nitrosopumilus (ammonia-oxidant), underlines how human cave activities did not impact these microbial traits. Besides, some bacterial taxa could be also considered as bioindicators of human presence being a result of contamination from the entrance 30,42 .
For instance, many human-related genera as, Lactobacillus (also as unique ASVs), Lactococcus, Legionella (only as unique ASVs), Staphylococcus and Streptococcus were mainly, or in some cases uniquely, found in show -caves. Similarly, the recurrence of opportunistic human pathogenic fungi such as Candida and dermatophytes (i.e. Trichosporon and Cutaneotrichosporon) throughout tourist caves, emphasize a probable direct mycobiome cave contamination as the result of the extensive human exploitation of these subterranean settings. Concerning microalgae, except for a widespread presence among caves of Ochrophyta phylum and goldbrown algae Chrysophyceae related class, the eukaryotic photosynthetic abundance background appeared patchy. Some caves were largely characterized by presence of particular classes, e.g. Bossea cave by Diatomea, Caudano by Dinophyceae and Trebouxiophyceae, Pertosa-Auletta by Chlorophyceae and Vento cave with Cryptophyceae and Trebouxiophyceae. However, the only notable differences were related to the relative abundance values and lack of unique ASVs of microalgal components harbored by the wild Costacalda cave, dominated by the class Chrysophyceae. This class is composed of taxa that prefer oligotrophic conditions and most species in this group show a mixotrophic metabolism, being able to shift between photosynthesis and ingesting smaller organisms or particles for food. We may hypothesize that the micro-algal component in pristine caves was based on a poorly biodiverse common core 18 , populated by species well adapted to cope with the typical challenges of the cave environment, such as strict oligotrophy and darkness 43 . The subsequent anthropization (artificial lighting and tourist flows) of these settings would have had a key role, both in importing allochthonous species and spreading the local and alien microalgal component even in cave's zones previously uncolonized 18,44 . Yet, the ASVs cores recorded in bacteria, archaea and algae indicate a high degree of adaptation and specialization for these microbial compartments in caves. Conversely, the narrow core observed for the fungal component may indicate a less strict adaptation to the cave environment and broad capacity to colonize different habitats, according to the high physiological, metabolic and stress-tolerance plasticity of these organisms.
In conclusion, this study provides for the first time a multi-spatial and extensive microbiota characterization of 5 Italian caves, among which 4 tourist and 1 pristine cave, shedding light on microbial diversity of different rapidly evolving subterranean environments. We highlighted how the 4 investigated show -caves share common microbial traits, by filtering microorganisms derived from the outside or human-related, multi-stress tolerant or capable of exploiting the extra supplies of organic matter or degradable compounds provided by tourist flows. Although human activities have been affecting these caves for a long time, the principal common microbial traits, related to the biogeochemical key processes, are still clearly detectable. Finally, we have provided some insights into the potential direct microbiome cave contamination by human-related microbial species. It is worth expanding this dataset by sampling additional underground environments, including both show-caves and wild caves, at local (Italian) geographic scales, to confirm the trends of microbial diversity pattern here found in anthropized caves with respect to the wild one. This study represents the groundwork for further microbiome cave analyses to shed light also on the functional guilds of each microbial component and their distribution and variation in both pristine and impacted settings.
Besides sharing the same karstic origin, the caves investigated, with the exception of Costacalda, are characterized by a long human exploitation background. Bossea was the first Italian show cave established in 1875 (https:// www. grott adibo ssea. com), while the Caudano cave was permanently opened to the public in 2002 (https:// grott edelc audano. com). Besides regular tourist flows, these caves have experienced exceptional human activity episodes over the last century, like the "700 h underground" experiment (Caudano cave in 1961), where 12 speleologists and some domestic animals lived inside the cave for 1 month, aiming to understand the effect of underground life on human and animals. In addition, Bossea cave has been the setting of many music and foodwine festivals. The Vento cave has been known since the seventeenth century and its cold air currents were used www.nature.com/scientificreports/ by local people as a natural cooler to preserve food b. The cave was later explored in the nineteenth century and opened to the public for the first time in 1967 (http:// grott adelv ento. com). The Pertosa-Auletta cave has been exploited by humans since the Neolithic, successively used during the Iron Age (the ruins of a stilt village were found), then converted into a Christian church during the twelfth century and into an air-raid shelter during WWII It was permanently turned into a tourist cave in the second half of the 1900s (https:// fonda zione mida. com/ grotte-perto sa-aulet ta). Costacalda is a natural (wild; i.e. not impacted) cave discovered in the spring of 2018, when it appeared as a hole about 10 cm wide on the ground. To date, the cave is only partially explored and entirely preserves its pristine structural condition (http:// www. spele ologi assi. it/ 82-somma ri).

Sampling design.
We applied an extensive sampling of sediments along the whole extension of each cave ( Fig. 8B) according to Piano et al. (2022) 20 , starting from the areas near the entrance, towards the deepest zones and at different distances from the tourist/speleological paths. A total of 12 in situ sampling sites were selected for the 4 show -caves, while 3 sites were selected in the Costacalda wild cave. For each sample, 9 technical sediment replicates subdivided into 3 triplets, up to 5 cm depth, were collected using sterile Falcon® tubes (50 ml), for a total of 459 samples. Samples were then stored in a cooler-bag until arrival at the laboratory, where the 3 replicates for each sample were pooled and homogenized. Twelve final samples were assembled for each show cave (Bossea, Caudano, del Vento and Pertosa-Auletta) and 3 for Costacalda, for a total of 51 samples. Sampling collection was carried out between June and September 2020.
Metagenomic DNA extraction and amplicon sequencing. Sediment samples were sieved, under sterile conditions, by removing coarse rock debris and metagenomic DNA was extracted from 0.5 g of sample using Qiagen DNeasy PowerSoil Pro Kit (Carlsbad, CA, USA). The Internal Transcribed Sequence 1 ribosomal region (ITS1), Eukaryotic SSU rRNA (18S) and hypervariable region V4 of 16S ribosomal gene were targeted to assess the fungal, general eukaryotic and prokaryotic community composition, respectively. The ITS1 region was amplified using barcoded primers ITS1F/ITS2, suitable for shorter read length 46 , the 18S region with the Euk_1391f./EukBr primers (http:// www. earth micro biome. org), while for the V4 region of 16S, barcoded F515/ www.nature.com/scientificreports/